Technical ISO Specification ISO/TS 12869-2 First edition Water quality Detection and 2024-04 quantification of Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction (qPCR) - Part 2: On-site methods Qualite de I'eau - Détection et quantification de Legionella spp. et/ou Legionella pneumophila par concentration et amplification génique par réaction de polymérisation en chaine quantitative (qPCR) - Partie 2: Méthodes sur site Referencenumber ISO/TS 12869-2:2024(en) @ISO2024 IS0/TS 12869-2:2024(en) COPYRIGHT PROTECTED DOCUMENT @ ISO 2024 All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or Iso's member body in the country ofthe requester. ISO copyright office CP 401 : Ch. de Blandonnet 8 CH-1214 Vernier, Geneva Phone:+41227490111 Email: [email protected] Website: www.iso.org Published in Switzerland @ IS0 2024 - All rights reserved ii IS0/TS 12869-2:2024(en) Contents Page Foreword. Introduction Scope. 1 2 .1 Normative references 3 Terms, definitions, symbols and abbreviated terms 2 .2 3.1 Terms and definitions 3.2 Symbols and abbreviated terms 3 Principle. 4 4 5 Sampling 6 General testing conditions 5 6.1 .5 General End users. 6.2 5 6.2.1 General 6.2.2 Manufacturer's instructions .5 6.2.3 Proficiency 6 Usability validation (human factors testing [HFTD) 6.2.4 .6 6.3 7 Premises 6.3.1 Manufacturerpremises End user premises 6.3.2 6.4 Apparatus and consumables (excluding reagents) 6.4.1 General 7 6.4.2 Safety 6.4.3 8 Concentration. 6.4.4 PCR (detection and quantification) 8 6.5 Reagents. 8 6.6 Decontamination of equipment and premises 9 6.7 Maintenance and calibration .9 Treatment and elimination of waste 6.8 9 Procedure. 7 9 7.1 9 Concentration 7.2 Bacterial concentration and bacterial recovery 9 7.2.1 General .9 7.2.2 Protocols 9 7.2.3 Stability of bacterial eluates and DNA 10 DNA amplification by PCR 7.3 10 7.3.1 10 General 7.3.2 Target sequences, primers and probes. 10 7.3.3 Amplification mix preparation. .10 7.4 Quantitative detection .11 7.4.1 .11 General Protocol 7.4.2 12 7.5 Qualitative detection 12 8 Expression of the results. .13 9 Technical protocol for the characterization and the validation of the method 13 9.1 13 General 9.2 Inclusivity and exclusivity of probes and primers, 13 9.3 Verification of the calibration function of the quantitative PCR phase 13 9.4 13 Verification of the PCR limit of quantification, LQqPCR 9.5 13 9.5.1 Principle. 13 9.5.2 Experimental design 14 @ IS0 2024 - All rights reserved iii